Recent elucidation of the fact that nitric oxide plays many biological roles has spurred special interest in this molecule. For instance, nitric oxide is believed to play a role in vasodilation. See Marletta et al., “Unraveling the biological significance of nitric oxide” Biofactors 2:219 (1990). Nitric oxide also appears to inhibit platelet aggregation by elevating intraplatelet levels of cyclic GMP. See Diodati et al, “Complexes of Nitric Oxide with Nucleophiles as Agents for the Controlled Biological Release of Nitric Oxide: Antiplatelet Effect” Thrombosis and Haemostasis 70:654 (1993)
More recently, nitric oxide is emerging as one of the main neurotransmitters in the central and peripheral nervous systems. See Snyder, “Janus faces of nitric oxide” Nature 364:577 (1993). It appears to play both neurotoxic roles, such as in AIDS dementia, and neuroprotective roles in degenerative problems such as Parkinson's and Huntington's diseases.
Given the growing importance of the molecule, there have been a number of attempts to develop means to measure cellular levels of nitric oxide. For example, a fiber optic nitric oxide chemiluminescent sensor has been developed. See Zhou and Arnold, “Response Characteristics and Mathematical Modeling for a Nitric Oxide Fiber-Optic Chemical Sensor” Anal. Chem. 68:1748 (1996). This sensor was constructed by holding a small amount of an internal reagent solution at the tip of a fiber-optic bundle with a piece of gas-permeable membrane. Nitric oxide diffuses across the membrane into this internal solution, where a chemiluminescent reaction between nitric oxide, hydrogen peroxide, and luminol takes place. The drawbacks of this sensor include the following: 1) the response time (approximately 8-17 seconds) is longer than the time needed for nitric oxide in the solution to be converted to nitrite; 2) the detection of nitric oxide is complicated by interferences from dopamine, uric acid, ascorbic acid, and cysteine, 3) the sensor is relatively large in size (greater than 6 mm in diameter) and thus difficult to use for the measurement of cellular nitric oxide levels (and impossible for intracellular measurements); and 4) the sensor has relatively poor sensitivity, i.e., a relatively high limit of detection (approximately 1.3 mM of nitric oxide).
Sensors involving sol-gel technology have also been attempted. The process involves hydrolyzing an alkoxide of silicon to produce a sol, which then undergoes polycondensation to form a gel. Biomolecules are immobilized by being entrapped in the sol-gel. In one case, horse-heart cytochrome c was encapsulated in a sol-gel and absorbance-based spectral shifts were used to monitor the binding of nitric oxide. See Blyth et al., “Sol-Gel Encapsulation of Metalloproteins for the Development of Optical Biosensors for Nitrogen Monoxide and Carbon Monoxide” Analyst 120:2725 (1995). Unfortunately, the sensor reaction is reported to have taken two hours to reverse, making dynamic measurements impossible.
What is needed is a sensor of relatively small size and good sensitivity that measures nitric oxide with little or no interference from other analytes in a short enough time period to permit dynamic measurements.